Effect of Doxycycline-Regulated Calnexin and Calreticulin Expression on Specific Thrombopoietin Productivity of Recombinant CHO Cells

Abstract

In an attempt to increase the specific TPO productivity (q_TPO) of rCHO cells (CHO-TPO), the effect of expression level of calnexin (CNX) and calreticulin (CRT) on q_TPO was investigated. To regulate the CNX and CRT expression level simultaneously, the Tet-Off system was first introduced in CHO-TPO cells and stable Tet-Off cells (TPO-Tet-Off) were screened by the luciferase assay. The rCHO cells with a doxycycline-regulated CNX and CRT expression system (TPO-CNX/CRT) were established by cotransfection of CNX and CRT expression vector and pTK-Hyg vector into TPO-Tet-Off cells and subsequent screening by Western blot analysis of CNX and CRT. The expression levels of CNX and CRT in TPO-CNX/CRT cells could be tightly regulated by adding different concentrations of doxycycline to a culture medium. Compared with the basal level (2 µg/mL doxycyline), a 2.9-fold increase in CNX expression and a 2.8-fold increase in CRT expression were obtained in the absence of doxycycline. This increased expression level of CNX and CRT resulted in a 1.9-fold increase in q_TPO without growth inhibition. Furthermore, in vivo biological activity of TPO was not changed by increasing the CNX and CRT expression level. Taken together, the results obtained here demonstrate that a simultaneous overexpression of CNX and CRT can increase the q_TPO of rCHO cells.